STATEMENT OF PROBLEM: Improving the probability of detecting invasive giant snakes is vital for the management of emerging or established populations. Detecting invasive species at low densities or prior to establishment is critical for successful control and eradication (Darling and Mahon 2011; Kolar et al. 2010; Lodge et al. 2006). Burmese pythons occupy thousands of square kilometers of mostly inaccessible habitats, and detection probabilities have been extremely low using traditional tools such as visual searching or trapping (Reed et al. 2011). These large constrictor snakes are detrimental to native species, especially imperiled or at risk species, such as wading birds. Tools for detection and control (e.g., detector dogs, remote sensing, attractant traps, "Judas snakes", etc.) have provided low detection with varying degrees of success for Burmese pythons in Florida.

Environmental DNA (eDNA) is increasingly being used for detection of non-native species to inform management actions, especially when traditional field methods are inadequate (Goldberg et al. 2011; Jerde et al. 2011; Mahon et al. 2013; Thomsen et al. 2012). Environmental DNA originates from cellular material shed by organisms (via skin, excrement, etc.) into water or soil, and can be used for species identification (Dejean et al. 2011; Thomsen et al. 2012). Environmental DNA methods have been shown to be time and cost effective in a number of systems (Goldberg et al. 2011; Jerde et al. 2011; Takahara et al. 2012) and may be preferable to traditional detection methods for constrictor snakes.

In a pilot study, eDNA methods detected radio tagged snakes in Collier County, and unsighted Burmese pythons in Everglades National Park and Deering Estates (Hunter et al. submitted). Further, water samples were collected in Holey Land Wildlife Management Area (HLWM) after an eyewitness report of a large snake, possibly an anaconda or Burmese python. From three collected samples, one resulted in a positive detection for Burmese pythons, while the tests for Anaconda were negative. The presence of pythons in HLWM and nearby Stormwater Treatment Area (STA) 5 indicates that Burmese pythons are moving further to the north and possibly approaching the ARM Loxahatchee National Wildlife Refuge boundaries.

The ARM Loxahatchee National Wildlife Refuge is an important area for native flora and fauna. The Refuge (also water conservation area (WCA) 1 is maintained to provide water storage and flood control in south Florida. The water storage areas, along with areas of Everglades National Park, are the remaining locations of the original Everglades habitat. The Refuge works to restore the Everglades ecosystem to the benefit of wildlife. Since Burmese pythons have the potential to significantly reduce native species, python detection and rapid removal prior to establishment could be of great benefit to the Refuge.

OBJECTIVES: Environmental DNA methods can assist with early detection of novel invasive species and range delimitation or expansion for established species. Further, these methods can be used to monitor habitat critical to the survival of imperiled native species and assess the success of rapid response control or removal efforts within strategic areas. Using the described eDNA methods we propose to (1) develop sampling density methodology for accurate detection of Burmese pythons in laboratory and field settings. (2) This information will then be used to develop sampling design in areas where Burmese pythons have not been identified to date. Detection limits and environmental variables that influence eDNA detection will be assessed. (2) Selected areas in and around ARM Loxahatchee National Wildlife Refuge will be surveyed at appropriate densities to help determine detection probabilities and range-boundaries of Burmese pythons. Samples will focus on Burmese python habitat within the south and west borders of the Refuge. Further, samples could be collected inside the Refuge at wading bird colonies possibly at risk of predation. Outside of the Refuge, sampling will focus on the adjoining areas where pythons have been detected, including WCAs, STAs and Everglades Agriculture Areas (EAAs).

METHODOLOGY: Species-specific quantitative PCR (qPCR) primers and probes have been developed for the established Burmese python, Northern African python,andboa constrictor, and two species of concern, the green and yellow anaconda (Hunter et al. submitted). Markers for the three established species were tested in laboratory trials and all five marker sets were tested on water samples collected at 21 sites throughout southern Florida and the Everglades. Burmese pythons were detected in 20 of 63 sampling events. Compared to traditional PCR primers, qPCR technology can greatly increase the precision, sensitivity, specificity and quantitative properties of detection (Hyatt et al. 2007; Phalen et al. 2011; Pilliod et al. 2013; Takahara et al. 2012; Thomsen et al. 2012; Wilcox et al. 2013). Specifically, the highly sensitive TaqMan (Applied Biosystems, Foster City, CA) qPCR method uses three species-specific markers (two primers and a fluorescently labelled probe) to detect the target DNA, and can be used to quantify the number of copies or relative amounts of targeted molecules in the sample. This information can be used to accurately estimate the likelihood of a species being present or absent in the environment, thus improving effective resource actions, making inferences about distribution and movement patterns, and developing long-term management strategies.

In phase 1 of the study, water collection in high density python populations over varying temporal and spatial scales will be used to determine the sampling scale needed for accurate detection. Quantification of minimum eDNA detection threshold and the influencing variables will be assessed. Detection information will be used to develop foundational detection probabilities and appropriate sampling schemes for 95% detection probabilities.

In phase 2, system level surveys will be conducted in and around ARM Loxahatchee National Wildlife Refuge to test for range limits and expansion into uninhabited areas. Using the previously determined sampling density, sample sites and sampling design will be determined to efficiently maximize python detections within the study budget. Areas include perimeter canals and levees along the south and west boundaries of the Refuge. Outside of the Refuge, areas with python detections, primary python habitat, and corridors leading to the Refuge will be targeted. Given the best available information, the areas to consider are STA1W and E, WCA 2A, and EAA boundaries adjoining the Refuge, specifically the L-7 canal and levee. Areas with previous sightings further from the Refuge boundary include STA5 and HLWM. Additionally, surveys of the other four species could be incorporated to obtain baseline data on their presence throughout the landscape.

With time and adequate resources, exploratory statistical models of site-occupancy will be extended to provide estimates of the probability of occurrence of eDNA molecules, the probability of false-negatives (given eDNA presence), and the probability of false-positives (given eDNA absence). Each of these probabilities will be specified as functions of covariates so that the effects of experimental treatments on each probability can be estimated directly.

WORK PLAN

FY 2014 Statistical sampling design development and start of Phase 1 field sampling in the Greater Everglades. Annual progress summary.

FY 2016 Completion of Phase 2 sampling and analysis of samples. With adequate information, time and resources, occupancy models will be development. Final progress summary and manuscript preparation. Throughout the course of data collection and model testing, experiments may need to be modified in order to improve site-occupancy models.

RELEVANCE AND BENEFITS: Environmental DNA has the potential to be a powerful tool for enhancing early detection of giant constrictor snakes for rapid response as well as providing a decision-support tool for long-term management strategies. Non-invasive monitoring of aquatic habitats can assist in identifying newly-colonized areas where pythons have not been detected, as well as movement corridors and pathways of dispersal. More precise information on the presence of harmful constrictors in critical habitats can inform spatiotemporal assessment of risk to imperiled native species (i.e., ground-dwelling birds, Florida panther), and potentially allow for targeted removal efforts prior to major ecological and economic impacts. Environmental DNA tools can also assist with short or long term monitoring to determining whether control or eradication efforts were successful.

COMMUNICATION PLAN, TECHNOLOGY and INFORMATION TRANSFER: Manuscripts will be prepared for publication as noted under the Work Plan and Products sections. A fact sheet will be developed and/or a project description will be posted to the SESC web site. Additionally, project results will be disseminated as presentations at scientific conferences and informal exchange of data with the funding agency and other research partners.

ANIMAL WELFARE: This project is operating under Approved IACUC #USGS/SESC 2013-04. All animal handling will be conducted in compliance with USDA APHIS standards and AWA regulations. All animals are handled using humane methods, meeting or exceeding conditions required by the Animal Welfare Act. Minimally invasive or post-mortem samples will be collected and approved methods of euthanasia that produce rapid unconsciousness will be used.